Toxoplasma gondii is an important human pathogen that has infected over 50 million people in the U.S., and over a billion people worldwide. This pathogen can cause severe disease in immunocompromised individuals (e.g., due to HIV/AIDS) and primary infections in pregnant women can cause death of the fetus. Toxoplasma is an obligate intracellular pathogen, and is capable of secreting effector proteins into the host cell that dramatically affect the severity of disease. To date, standard proteomics approaches have not allowed for a comprehensive identification of Toxoplasma proteins that are secreted from the parasite in vivo since they represent only a small fraction of host cell protein content. Using a novel approach to comprehensively analyze Toxoplasma proteins secreted in vivo, particularly those proteins that are secreted into the host cell, we aim to identify new classes of host-interacting proteins that play significant roles in Toxoplasma pathogenesis. To do this we have genetically engineered Toxoplasma to express a mutant methionyl tRNA synthetase (MetRS) that is capable of charging endogenous Met-tRNAs with a methionine analog that can be tagged using click chemistry cycloaddition reactions, allowing for subsequent purification from the complex milieu of the host cell. Preliminary data show that Toxoplasma strains expressing this mutant MetRS readily incorporate the methionine analog into nascent proteins, while wild type strains do not. This approach will be used to 1) qualitatively analyze temporal changes in the secretome during the parasite growth cycle and 2) selectively purify and identify proteins secreted in vivo using mass spectrometry. These studies will provide answers to fundamental questions regarding the complexity of the Toxoplasma secretome during infection and when they are secreted. Moreover, given the important role that secreted proteins play in all intracellular pathogens, including Toxoplasma, we hope to identify previously uncharacterized effector proteins that play significant roles in parasite invasion, growth, intracellular survival, and virulence. Such effectors may represent new potential targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a human pathogen that causes severe disease in immunocompromised individuals and in the developing fetus, and has infected over 50 million people in the U.S. The work described in this proposal aims to identify Toxoplasma gene products that may play a significant role in causing severe disease using a novel approach to comprehensively identify proteins that are secreted from the parasite into the host cell. These gene products may play important roles in Toxoplasma biology and represent new therapeutic targets for combating this important human pathogen.